Dissecting the genetic determinants of bacterial DNA degradation by bacteriophage T5

Upon infection of Escherichia coli, the virulent bacteriophage T5 employs a unique two-step mechanism to transfer its 122-kb genome: only 8% of the DNA are initially transferred, allowing expression of pre-early genes that alter host functions before the remaining DNA is delivered. Early infection triggers rapid host DNA degradation and nucleotide catabolism, processes partly controlled by the pre-early genes encoding the predicted metallo-phosphatase A1 and the dNMP phosphatase Dmp. However, the functions of most of the 17 proteins encoded by the first-step transfer DNA (FST-DNA) remain unknown. Using reverse genetics, we engineered several T5 mutants carrying deletions in the FST-DNA. One of them carries only four pre-early genes (A1, A2, hdi and 009), demonstrating that thirteen of the 17 predicted genes are dispensable under laboratory conditions. Mutant characterization showed that only A1 and the predicted DNA-binding protein gene A2 are essential for productive infection, while dmp enhances phage virulence. Notably, seven pre-early genes (dmp, A1, hdi, hegG, 011, 013 and 015) proved toxic when expressed in E. coli without other viral factors, causing severe morphological changes or nucleoid disorganization, while one gene compromised membrane integrity. The essential gene A1, which is conserved among all viruses in the Demerecviridae family, emerged as the primary driver of host genome degradation, both essential and sufficient for chromosomal DNA digest in vivo. These findings advance our understanding of how T5 manipulates its host during the critical early stages of infection, offering new insights into phage-host interactions and the molecular strategies viruses use to subvert bacterial cells.